Cotton event mon 88913 and compositions and methods for detection thereof

ABSTRACT

The present invention provides a cotton plant event MON 88913 compositions and seed. Also provided are assays for detecting the presence of the cotton plant event MON 88913 based on a DNA sequence and the use of this DNA sequence as a molecular marker in a DNA detection method.

This application claims benefit of U.S. Provisional Application No.60/447,184, filed Feb. 12, 2003, the entire contents of which areincorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to the field of plant molecular biology.More specifically, the invention relates to a glyphosate tolerant cottonevent MON 88913 and to assays and methods for detecting the presence ofcotton event MON 88913 DNA in a plant sample and compositions thereof.

BACKGROUND OF THE INVENTION

Cotton is an important fiber crop in many areas of the world. Themethods of biotechnology have been applied to cotton for improvement ofthe agronomic traits and the quality of the product. The method ofintroducing transgenes into cotton plants has been demonstrated in U.S.Pat. No. 5,004,863. One such agronomic trait important in cottonproduction is herbicide tolerance, in particular, tolerance toglyphosate herbicide. This trait has been introduced into cotton plantsand is a successful product now used in cotton production. The currentcommercial Roundup Ready® cotton event (1445) provides excellenttolerance to glyphosate, the active ingredient in Roundup®, through thefour-leaf stage (Nida et al., J. Agric. Food Chem. 44:1960-1966, 1996;Nida et al., J. Agric. Food Chem. 44:1967-1974, 1996). However, foliarapplication beyond the four-leaf stage must be limited due toinsufficient tolerance in male reproductive tissues in certainenvironmental conditions. This lack of male reproductive toleranceappears to be a result of insufficient CP4 EPSPS expression in criticaltissues, higher sensitivity of these tissues to glyphosate, andaccumulation of high amounts of glyphosate in these strong sink tissues(Pline et al., Weed Sci. 50:438-447, 2002). There is a need for a cottonplant more highly glyphosate tolerant than Roundup Ready® cotton 1445.

It would be advantageous to be able to detect the presence of aparticular event in order to determine whether the progeny of a sexualcross contain a transgene of interest. In addition, a method fordetecting a particular event would be helpful for complying withregulations requiring pre-market approval or labeling of foods derivedfrom recombinant crop plants, for example. It is possible to detect thepresence of a transgene by any well known nucleic acid detection methodsuch as the polymerase chain reaction (PCR) or DNA hybridization usingnucleic acid probes. These detection methods generally focus onfrequently used genetic elements, such as promoters, 3′ transcriptionterminators, marker genes, etc. As a result, such methods may not beuseful for discriminating between different events, particularly thoseproduced using the same DNA construct unless the sequence of genomicchromosomal DNA adjacent to the inserted DNA (“flanking genomic DNA”) isknown. Event-specific DNA detection methods for a glyphosate tolerantcotton event 1445 have been described (US 20020120964, hereinincorporated by reference in its entirety).

The present invention relates to a glyphosate tolerant cotton event MON88913, compositions contained therein, and to the method for thedetection of the transgene/genomic insertion region in cotton event MON88913 and progeny thereof.

SUMMARY OF THE INVENTION

The present invention is related to the transgenic cotton eventdesignated MON 88913 having seed deposited with American Type CultureCollection (ATCC) with Accession No. PTA-4854. Another aspect of theinvention comprises the progeny plants, or seeds, or regenerable partsof the plants and seeds of the cotton event MON 88913. The inventionalso includes plant parts of cotton event MON 88913 that include, butare not limited to pollen, ovule, flowers, bolls, lint, shoots, roots,and leaves. The invention relates to a cotton plant having a glyphosatetolerant phenotype and the novel genetic compositions of MON 88913.

One aspect of the invention provides DNA compositions and methods fordetecting the presence of a transgene/genomic junction region fromcotton plant event MON 88913. Isolated DNA molecules are provided thatcomprise at least one transgene/genomic junction DNA molecule selectedfrom the group consisting of SEQ ID NO:1 and SEQ ID NO:2, andcomplements thereof, wherein the junction molecule spans the insertionsite that comprises a heterologous DNA inserted into the cotton genomeand the genomic DNA from the cotton cell flanking the insertion site incotton event MON 88913. A cottonseed and plant material thereofcomprising these molecules is an aspect of this invention.

An isolated novel DNA molecule is provided that is a 5′transgene/genomic region SEQ ID NO:3 or the complement thereof, whereinthis DNA molecule is novel in cotton event MON 88913. A cotton plant andseed comprising SEQ ID NO:3 in its genome is an aspect of thisinvention. According to another aspect of the invention, an isolated DNAmolecule is provided that is a 3′ transgene/genomic region SEQ ID NO:4,or the complement thereof, wherein this DNA molecule is novel in cottonevent MON 88913. A cotton plant and seed comprising SEQ ID NO:4 in itsgenome is an aspect of this invention.

According to another aspect of the invention, two DNA molecules areprovided for use in a DNA amplification method, wherein the first DNAmolecule comprises at least 11 or more contiguous polynucleotides of anyportion of the transgene region of the DNA molecule of SEQ ID NO:3 and aDNA molecule of similar length of any portion of a 5′ flanking cottongenomic DNA region of SEQ ID NO:3, where these DNA molecules when usedtogether are useful as a DNA primer set in a DNA amplification methodthat produces an amplicon. The amplicon produced using the DNA primerset in the DNA amplification method is diagnostic for cotton event MON88913. Any amplicon produced from MON 88913 DNA by DNA primers that arehomologous or complementary to any portion of SEQ ID NO:3 is an aspectof the invention.

According to another aspect of the invention, two DNA molecules areprovided for use in a DNA amplification method, wherein the first DNAmolecule comprises at least 11 or more contiguous polynucleotides of anyportion of the transgene region of the DNA molecule of SEQ ID NO:4 and aDNA molecule of similar length of any portion of a 3′ flanking cottongenomic DNA of SEQ ID NO:4, where these DNA molecules are useful as aDNA primer set in a DNA amplification method. The amplicon producedusing the DNA primer set in the DNA amplification method is diagnosticfor cotton event MON 88913. The amplicons produced from MON 88913 DNA byDNA primers that are homologous or complementary to any portion of SEQID NO:4 are an aspect of the invention.

According to another aspect of the invention, methods of detecting thepresence of DNA corresponding specifically to the cotton event MON 88913DNA in a sample are provided. Such methods comprise: (a) contacting thesample comprising DNA with a DNA primer set that, when used in a nucleicacid amplification reaction with genomic DNA from cotton event MON 88913produces an amplicon that is diagnostic for cotton event MON 88913 (b)performing a nucleic acid amplification reaction, thereby producing theamplicon; and (c) detecting the amplicon.

According to another aspect of the invention, methods of detecting thepresence of DNA corresponding specifically to the cotton event MON 88913DNA in a sample are provided. Such methods comprising: (a) contactingthe sample comprising DNA with a DNA probe comprising SEQ ID NO:1 or SEQID NO:2, that hybridize under stringent hybridization conditions withgenomic DNA from cotton event MON 88913 and does not hybridize under thestringent hybridization conditions with a control cotton plant DNA; (b)subjecting the sample and probe to stringent hybridization conditions;and (c) detecting hybridization of the probe to the cotton event MON88913 DNA.

According to another aspect of the invention, methods of producing acotton plant that tolerates application of glyphosate are provided thatcomprise the steps of (a) sexually crossing a first parental cottonevent MON 88913 comprising the expression cassettes of the presentinvention, which confers tolerance to application of glyphosate, and asecond parental cotton plant that lacks the glyphosate tolerance,thereby producing a plurality of progeny plants; and (b) selecting aprogeny plant that tolerates application of glyphosate. Such methods mayoptionally comprise the further step of backcrossing the progeny plantto the second parental cotton plant and selecting for glyphosatetolerant progeny to produce a true-breeding cotton variety thattolerates application of glyphosate.

According to another aspect of the invention, a method is provided fordetermining the zygosity of the progeny of cotton event MON 88913comprising: (a) contacting the sample comprising cotton DNA with aprimer set comprising SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, and SEQ ID NO:25 that when used in a nucleic-acid amplificationreaction with genomic DNA from cotton event MON 88913, produces a firstamplicon that is diagnostic for cotton event MON 88913; and (b)performing a nucleic acid amplification reaction, thereby producing thefirst amplicon; and (c) and detecting the first amplicon; and (d)contacting the sample comprising cotton DNA with said primer set, thatwhen used in a nucleic-acid amplification reaction with genomic DNA fromcotton plants produces a second amplicon comprising the native cottongenomic DNA homologous to the cotton genomic region of a transgeneinsertion identified as cotton event MON 88913; and (e) performing anucleic acid amplification reaction, thereby producing the secondamplicon; and (0 and detecting the second amplicon; and (g) comparingthe first and second amplicons in a sample, wherein the presence of bothamplicons indicates the sample is heterozygous for the transgeneinsertion.

A method for determining zygosity comprising contacting a cotton DNAsample with using with primers and probes comprising SEQ ID NO:21, SEQID NO:22, SEQ ID NO:23, SEQ ID NO:24, and SEQ ID NO:25; using anendpoint Taqman® PCR condition; and detecting the amplicon products.

A method for controlling weeds in a crop or field of cotton event MON88913 comprising the step of applying a herbicidally effective amount ofglyphosate containing herbicide to the field of MON 88913 cotton.

The foregoing and other aspects of the invention will become moreapparent from the following detailed description and accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Plasmid map of pMON51915.

FIG. 2. Genomic organization of insert in cotton event MON 88913.

FIG. 3. MON 88913 5′ DNA junction sequence (SEQ ID NO:1) and 3′ DNAjunction sequence (SEQ ID NO:2).

FIG. 4. MON 88913 5′ transgene/genomic DNA region (SEQ ID NO:3).

FIG. 5. MON 88913 3′ transgene/genomic DNA region (SEQ ID NO:4).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to a glyphosate tolerant cotton event MON88913, compositions contained therein, and to the method for thedetection of the transgene/genomic insertion region in cotton event MON88913 and progeny thereof. The following definitions and methods areprovided to better define the present invention and to guide those ofordinary skill in the art in the practice of the present invention.Unless otherwise noted, terms and abbreviations are to be understoodaccording to conventional usage by those of ordinary skill in therelevant art. Definitions of common terms in molecular biology may alsobe found in Rieger et al., Glossary of Genetics: Classical andMolecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin,Genes V, Oxford University Press: New York, 1994. The nomenclature forDNA bases as set forth at 37 CFR §1.822 is used.

As used herein, the term “cotton” means Gossypium hirsutum and includesall plant varieties that can be bred with cotton event MON 88913. Theplant of the present invention is a cotton plant, more specifically thecotton plant MON 88913.

As used herein, the term “comprising” means “including but not limitedto”.

As used herein, the term “crop” refers to cultivated plants or parts ofplants, such as are grown in a field, plot, row, greenhouse, flat, orcontainer.

“Glyphosate” refers to N-phosphonomethylglycine and its salts.N-phosphonomethylglycine is a well-known herbicide that has activity ona broad spectrum of plant species. Glyphosate is the active ingredientof Roundup® (Monsanto Co.), a safe herbicide having a desirably shorthalf-life in the environment. Glyphosate is the active ingredient ofRoundup® herbicide (Monsanto Co.). Treatments with “glyphosateherbicide” refer to treatments with the Roundup®, Roundup Ultra®,Roundup Pro® herbicide or any other herbicide formulation containingglyphosate. Examples of commercial formulations of glyphosate include,without restriction, those sold by Monsanto Company as ROUNDUP®,ROUNDUP® ULTRA, ROUNDUP® ULTRAMAX, ROUNDUP® WEATHERMAX, ROUNDUP® CT,ROUNDUP® EXTRA, ROUNDUP® BIACTIVE, ROUNDUP® BIOFORCE, RODEO®, POLARIS®,SPARK® and ACCORD® herbicides, all of which contain glyphosate as itsisopropylammonium salt; those sold by Monsanto Company as ROUNDUP® DRYand RIVAL® herbicides, which contain glyphosate as its ammonium salt;that sold by Monsanto Company as ROUNDUP® GEOFORCE, which containsglyphosate as its sodium salt; and that sold by Syngenta Crop Protectionas TOUCHDOWN® herbicide, which contains glyphosate as itstrimethylsulfonium salt. When applied to a plant surface, glyphosatemoves systemically through the plant. Glyphosate is phytotoxic due toits inhibition of the shikimic acid pathway, which provides a precursorfor the synthesis of aromatic amino acids. Glyphosate inhibits theenzyme 5-enolpyruvyl-3-phosphoshikimate synthase (EPSPS) found inplants. Glyphosate tolerance can be achieved by the expression ofbacterial EPSPS variants and plant EPSPS variants that have loweraffinity for glyphosate and therefore retain their catalytic activity inthe presence of glyphosate (U.S. Pat. Nos. 5,633,435, 5,094,945,4,535,060, and 6,040,497).

A transgenic “event” is produced by transformation of a plant cell withheterologous DNA, e.g., a nucleic acid construct (pMON51915, FIG. 1)that includes a transgene of interest; regeneration of a population ofplants resulting from the insertion of the transgene into the genome ofthe plant cell, and selection of a particular plant characterized byinsertion into a particular genome location. The term “event” refers tothe original transformant plant and progeny of the transformant thatinclude the heterologous DNA. The term “event” also includes progenyproduced by a sexual outcross between the event and another plant thatwherein the progeny includes the heterologous DNA. Even after repeatedback-crossing to a recurrent parent, the inserted DNA and flankinggenomic DNA from the transformed parent event is present in the progenyof the cross at the same chromosomal location. The term “event” alsorefers to DNA from the original transformant comprising the insertedDNA, and flanking genomic sequence immediately adjacent to the insertedDNA, that would be expected to be transferred to a progeny that receivesthe inserted DNA including the transgene of interest as the result of asexual cross of one parental line that includes the inserted DNA (e.g.,the original transformant and progeny resulting from selfing) and aparental line that does not contain the inserted DNA.

The expression of foreign genes in plants is known to be influenced bytheir chromosomal position, perhaps due to chromatin structure (e.g.,heterochromatin) or the proximity of transcriptional regulation elements(e.g., enhancers) close to the integration site (Weising et al., Ann.Rev. Genet. 22:421-477, 1988). For this reason, it is often necessary toscreen a large number of events in order to identify an eventcharacterized by optimal expression of a introduced gene of interest.For example, it has been observed in plants and in other organisms thatthere may be a wide variation in levels of expression of an introducedtransgene among events. There may also be differences in spatial ortemporal patterns of expression, for example, differences in therelative expression of a transgene in various plant tissues, that maynot correspond to the patterns expected from transcriptional regulatoryelements present in the introduced gene construct. For this reason, itis common to produce hundreds to thousands of different events andscreen those events for a single event that has desired transgeneexpression levels and patterns for commercial purposes. An event thathas desired levels or patterns of transgene expression is useful forintrogressing the transgene into other genetic backgrounds by sexualcrossing using conventional breeding methods. Progeny of such crossesmaintain the transgene expression characteristics of the originaltransformant. This strategy is used to ensure reliable gene expressionin a number of varieties that are well adapted to local growingconditions and market demands.

A glyphosate tolerant cotton plant can be bred by first sexuallycrossing a first parental cotton plant, consisting of a cotton plantgrown from the transgenic cotton plant cell derived from transformationwith the plant expression cassettes contained in pMON51915 and thattolerates application of glyphosate herbicide, with a second parentalcotton plant that lacks the tolerance to glyphosate herbicide, therebyproducing a plurality of first progeny plants; and then selecting afirst progeny plant that is tolerant to glyphosate herbicide; andselfing the first progeny plant, thereby producing a plurality of secondprogeny plants; and then selecting from the second progeny plants aglyphosate herbicide tolerant plant. These steps can further include theback-crossing of the first glyphosate tolerant progeny plant or thesecond glyphosate tolerant progeny plant to the second parental cottonplant or a third parental cotton plant, thereby producing a cotton plantthat tolerates the application of glyphosate herbicide. In the presentinvention, the transgenic cotton plant is also defined as cotton eventMON 88913 and may be referred to herein as MON 88913.

It is also to be understood that two different transgenic plants canalso be mated to produce offspring that contain two independentlysegregating added, exogenous genes. Selfing of appropriate progeny canproduce plants that are homozygous for both added, exogenous genes.Back-crossing to a parental plant and out-crossing with a non-transgenicplant are also contemplated, as is vegetative propagation. Descriptionsof other breeding methods that are commonly used for different traitsand crops can be found in one of several references, e.g., Fehr, inBreeding Methods for Cultivar Development, Wilcox J. ed., AmericanSociety of Agronomy, Madison Wis. (1987).

A “probe” is an isolated nucleic acid to which is attached aconventional detectable label or reporter molecule, e.g., a radioactiveisotope, ligand, chemiluminescent agent, or enzyme. Such a probe iscomplementary to a strand of a target nucleic acid, in the case of thepresent invention, to a strand of genomic DNA from MON 88913 whetherfrom a MON 88913 plant or from a sample that includes MON 88913 DNA.Probes according to the present invention include not onlydeoxyribonucleic or ribonucleic acids but also polyamides and otherprobe materials that bind specifically to a target DNA sequence and canbe used to detect the presence of that target DNA sequence.

DNA primers are isolated polynucleic acids that are annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, then extended alongthe target DNA strand by a polymerase, e.g., a DNA polymerase. A DNAprimer pair or a DNA primer set of the present invention refer to atleast two DNA primer molecules useful for amplification of a targetnucleic acid sequence, e.g., by the polymerase chain reaction (PCR) orother conventional nucleic acid amplification methods.

Probes and primers are generally 11 polynucleotides or more in length,often 18 polynucleotides or more, 24 polynucleotides or more, or 30polynucleotides or more. Such probes and primers are selected to be ofsufficient length to hybridize specifically to a target sequence underhigh stringency hybridization conditions. Preferably, probes and primersaccording to the present invention have complete sequence similaritywith the target sequence, although probes differing from the targetsequence that retain the ability to hybridize to target sequences may bedesigned by conventional methods.

Methods for preparing and using probes and primers are described, forexample, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3,ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989 (hereinafter, “Sambrook et al., 1989”); CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates)(hereinafter, “Ausubel et al., 1992”); and Innis et al., PCR Protocols:A Guide to Methods and Applications, Academic Press: San Diego, 1990.PCR DNA primer pairs can be derived from a known sequence, for example,by using computer programs intended for that purpose such as Primer(Version 0.5, 1991, Whitehead Institute for Biomedical Research,Cambridge, Mass.).

Primers and probes based on the flanking genomic DNA and transgeneinsert sequences disclosed herein can be used to confirm (and, ifnecessary, to correct) the disclosed DNA sequences by conventionalmethods, e.g., by isolation of genomic DNA from MON 88913, re-cloningthe transgene/genomic regions and sequencing such DNA molecules.

The nucleic acid probes and primers of the present invention hybridizeunder stringent conditions to a target DNA molecule. Any conventionalnucleic acid hybridization or amplification method can be used toidentify the presence of DNA from a transgenic event in a sample.Polynucleic acid molecules or fragments thereof are capable ofspecifically hybridizing to other nucleic acid molecules under certaincircumstances. As used herein, two polynucleic acid molecules are saidto be capable of specifically hybridizing to one another if the twomolecules are capable of forming an anti-parallel, double-strandednucleic acid structure. A nucleic acid molecule is said to be the“complement” of another nucleic acid molecule if they exhibit completecomplementarity. As used herein, molecules are said to exhibit “completecomplementarity” when every nucleotide of one of the molecules iscomplementary to a nucleotide of the other. Two molecules are said to be“minimally complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder at least conventional “low-stringency” conditions. Similarly, themolecules are said to be “complementary” if they can hybridize to oneanother with sufficient stability to permit them to remain annealed toone another under conventional “high-stringency” conditions.Conventional stringency conditions are described by Sambrook et al.,1989, and by Haymes et al., In: Nucleic Acid Hybridization, A PracticalApproach, IRL Press, Washington, D.C. (1985), Departures from completecomplementarity are therefore permissible, as long as such departures donot completely preclude the capacity of the molecules to form adouble-stranded structure. In order for a nucleic acid molecule to serveas a primer or probe it need only be sufficiently complementary insequence to be able to form a stable double-stranded structure under theparticular solvent and salt concentrations employed.

As used herein, a substantially homologous DNA sequence is the sequenceof a DNA molecule that will specifically hybridize to the complement ofa target DNA molecule to which it is being compared under highstringency conditions. Appropriate stringency conditions that promoteDNA hybridization, for example, 6.0× sodium chloride/sodium citrate(SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C., areknown to those skilled in the art or can be found in Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Forexample, the salt concentration in the wash step can be selected from alow stringency of about 2.0×SSC at 50° C. to a high stringency of about0.2×SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to high stringency conditions at about 65° C. Both temperature andsalt may be varied, or either the temperature or the salt concentrationmay be held constant while the other variable is changed. In a preferredembodiment, a polynucleic acid of the present invention willspecifically hybridize to one or more of the nucleic acid molecules setforth in SEQ ID NO: 3 or 4, or complements thereof or fragments ofeither under moderately stringent conditions, for example at about2.0×SSC and about 65° C. In a particularly preferred embodiment, anucleic acid of the present invention will specifically hybridize to oneor more of the nucleic acid molecules set forth in SEQ ID NO:3 or 4 orcomplements or fragments of either under high stringency conditions. Inone aspect of the present invention, a preferred marker nucleic acidmolecule of the present invention has the nucleic acid sequence setforth in SEQ ID NO:1 or 2 or complements thereof or fragments of either.In another aspect of the present invention, a preferred marker nucleicacid molecule of the present invention shares a substantial portion ofits sequence identity with the nucleic acid sequence set forth in SEQ IDNO:1 or SEQ ID NO:2 or complement thereof or fragments of either,wherein the sequence identity is between 80% and 100% or 90% and 100%.In a further aspect of the present invention, a preferred marker nucleicacid molecule of the present invention shares between 95% and 100%sequence identity with the sequence set forth in SEQ ID NO:1 or SEQ IDNO:2 or complement thereof or fragments of either. SEQ ID NO:1 or SEQ IDNO:2 may be used as markers in plant breeding methods to identify theprogeny of genetic crosses similar to the methods described for simplesequence repeat DNA marker analysis, in “DNA markers: Protocols,applications, and overviews: (1997) 173-185, Cregan, et al., eds.,Wiley-Liss NY; herein incorporated by reference in its entirely. Thehybridization of the probe to the target DNA molecule can be detected byany number of methods known to those skilled in the art, these caninclude, but are not limited to, fluorescent tags, radioactive tags,antibody based tags, and chemiluminescent tags.

Regarding the amplification of a target nucleic acid sequence (e.g., byPCR) using a particular amplification primer pair, “stringentconditions” are conditions that permit the primer pair to hybridize onlyto the target nucleic acid sequence to which a primer having thecorresponding wild-type sequence (or its complement) would bind andpreferably to produce a unique amplification product, the amplicon, in aDNA thermal amplification reaction.

The term “specific for (a target sequence)” indicates that a probe orprimer hybridizes under stringent hybridization conditions only to thetarget sequence in a sample comprising the target sequence.

As used herein, “amplified DNA” or “amplicon” refers to the product ofpolynucleic acid amplification method directed to a target polynucleicacid molecule that is part of a polynucleic acid template. For example,to determine whether a cotton plant resulting from a sexual crosscontains transgenic event genomic DNA from the cotton event MON 88913plant of the present invention, DNA that is extracted from a cottonplant tissue sample may be subjected to a polynucleic acid amplificationmethod using a primer pair that includes a primer derived from DNAsequence in the genome of the MON 88913 plant adjacent to the insertionsite of the inserted heterologous DNA (transgene DNA), and a secondprimer derived from the inserted heterologous DNA to produce an ampliconthat is diagnostic for the presence of the MON 88913 event DNA. Thediagnostic amplicon is of a length and has a DNA sequence that is alsodiagnostic for the event. The amplicon may range in length from thecombined length of the primer pairs plus one nucleotide base pair,preferably plus about fifty nucleotide base pairs (bps), more preferablyplus about two hundred-fifty nucleotide base pairs, and even morepreferably plus about four hundred-fifty nucleotide base pairs or more.Alternatively, a primer pair can be derived from genomic sequence onboth sides of the inserted heterologous DNA so as to produce an ampliconthat includes the entire insert polynucleotide sequence (e.g., a forwardprimer isolated from the genomic portion of SEQ ID NO:3 and a reverseprimer isolated from the genomic portion of SEQ ID NO:4 that amplifies aDNA molecule comprising the two expression cassettes of pMON51915 DNAfragment that was inserted into the MON 88913 genome, the insertcomprising about 8,512 bps of the insert, FIG. 2). A member of a primerpair derived from the plant genomic sequence may be located a distancefrom the inserted DNA sequence, this distance can range from onenucleotide base pair up to about twenty thousand nucleotide base pairs.The use of the term “amplicon” specifically excludes primer dimers thatmay be formed in the DNA thermal amplification reaction.

Polynucleic acid amplification can be accomplished by any of the variouspolynucleic acid amplification methods known in the art, including thepolymerase chain reaction (PCR). Amplification methods are known in theart and are described, inter alia, in U.S. Pat. Nos. 4,683,195 and4,683,202 and in PCR Protocols: A Guide to Methods and Applications, ed.Innis et al., Academic Press, San Diego, 1990. PCR amplification methodshave been developed to amplify up to 22 kb (kilobase) of genomic DNA andup to 42 kb of bacteriophage DNA (Cheng et al., Proc. Natl. Acad. Sci.USA 91:5695-5699, 1994). These methods as well as other methods known inthe art of DNA amplification may be used in the practice of the presentinvention. The sequence of the heterologous DNA insert or flankinggenomic DNA sequence from MON 88913 can be verified (and corrected ifnecessary) by amplifying such DNA molecules from the MON 88913 seed orplants grown from the seed deposited with the ATCC having accession no.PTA-4854, using primers derived from the sequences provided herein,followed by standard DNA sequencing of the PCR amplicon or cloned DNAfragments thereof. DNA detection kits that are based on DNAamplification methods contain DNA primers that specifically amplify adiagnostic amplicon. The kit may provide an agarose gel based detectionmethod, endpoint Taqman®, or any number of methods of detecting thediagnostic amplicon that are known in the art. A kit that contains DNAprimers that are homologous or complementary to any portion of SEQ IDNO:3 or SEQ ID NO:4 is an object of the invention.

The amplicon produced by these methods may be detected by a plurality oftechniques. One such method is Genetic Bit Analysis (Nikiforov, et al.Nucleic Acid Res. 22:4167-4175, 1994) where a DNA oligonucleotide isdesigned that overlaps both the adjacent flanking genomic DNA sequenceand the inserted DNA sequence. The oligonucleotide is immobilized inwells of a microtiter plate. Following PCR of the region of interest(using one primer in the inserted sequence and one in the adjacentflanking genomic sequence), a single-stranded PCR product can behybridized to the immobilized oligonucleotide and serve as a templatefor a single base extension reaction using a DNA polymerase and labelleddideoxynucleotide triphosphates (ddNTPs) specific for the expected nextbase. Readout may be fluorescent or ELISA-based. A signal indicatespresence of the transgene/genomic sequence due to successfulamplification, hybridization, and single base extension.

Another method is the Pyrosequencing technique as described by Winge(Innov. Pharma. Tech. 00:18-24, 2000). In this method an oligonucleotideis designed that overlaps the adjacent genomic DNA and insert DNAjunction. The oligonucleotide is hybridized to single-stranded PCRproduct from the region of interest (one primer in the inserted sequenceand one in the flanking genomic sequence) and incubated in the presenceof a DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5′phosphosulfate and luciferin. Deoxynucleotide triphosphates (dNTPs) areadded individually and the incorporation results in a light signal thatis measured. A light signal indicates the presence of thetransgene/genomic sequence due to successful amplification,hybridization, and single or multi-base extension.

Fluorescence Polarization as described by Chen, et al., (Genome Res.9:492-498, 1999) is a method that can be used to detect the amplicon ofthe present invention. Using this method an oligonucleotide is designedwhich overlaps the genomic flanking and inserted DNA junction. Theoligonucleotide is hybridized to single-stranded PCR product from theregion of interest (one primer in the inserted DNA and one in theflanking genomic DNA sequence) and incubated in the presence of a DNApolymerase and a fluorescent-labeled ddNTP. Single base extensionresults in incorporation of the ddNTP. Incorporation can be measured asa change in polarization using a fluorometer. A change in polarizationindicates the presence of the transgene/genomic sequence due tosuccessful amplification, hybridization, and single base extension.

Taqman® (PE Applied Biosystems, Foster City, Calif.) is described as amethod of detecting and quantifying the presence of a DNA sequence andis fully understood in the instructions provided by the manufacturer.Briefly, a FRET oligonucleotide probe is designed which overlaps thegenomic flanking and insert DNA junction. The FRET probe and PCR primers(one primer in the insert DNA sequence and one in the flanking genomicsequence) are cycled in the presence of a thermostable polymerase anddNTPs. Hybridization of the FRET probe results in cleavage and releaseof the fluorescent moiety away from the quenching moiety on the FRETprobe. A fluorescent signal indicates the presence of thetransgene/genomic sequence due to successful amplification andhybridization.

Molecular Beacons have been described for use in sequence detection asdescribed in Tyangi, et al. (Nature Biotech. 14:303-308, 1996) Briefly,a FRET oligonucleotide probe is designed that overlaps the flankinggenomic and insert DNA junction. The unique structure of the FRET proberesults in it containing secondary structure that keeps the fluorescentand quenching moieties in close proximity. The FRET probe and PCRprimers (one primer in the insert DNA sequence and one in the flankinggenomic sequence) are cycled in the presence of a thermostablepolymerase and dNTPs. Following successful PCR amplification,hybridization of the FRET probe to the target sequence results in theremoval of the probe secondary structure and spatial separation of thefluorescent and quenching moieties. A fluorescent signal results. Afluorescent signal indicates the presence of the flanking/transgeneinsert sequence due to successful amplification and hybridization.

DNA detection kits can be developed using the compositions disclosedherein and the methods well known in the art of DNA detection. The kitsare useful for identification of cotton event MON 88913 DNA in a sampleand can be applied to methods for breeding cotton plants containing MON88913 DNA. The kits contain DNA sequences that are useful as primers orprobes and that are homologous or complementary to any portion of SEQ IDNO:3 or SEQ ID NO:4 or to DNA sequences homologous or complementary toDNA contained in any of the transgene genetic elements of pMON51915 thathave been inserted into MON 88913 DNA (FIG. 2). These DNA sequences canbe used in DNA amplification methods (PCR) or as probes in polynucleicacid hybridization methods, i.e., Southern analysis, northern analysis.The transgene genetic elements contained in MON 88913 DNA (FIG. 2)include a first expression cassette comprising the Figwort mosaicpromoter constructed as a chimeric promoter element with the Arabidopsiselongation factor 1-alpha (At.Eflα) promoter (FMV35S/Eflα, U.S. Pat. No.6,462,258, SEQ ID NO:28, herein incorporated by reference in itsentirely), operably linked to the Arabidopsis elongation factor 1-alphatranslational leader and intron (Genbank accession number X16430 asdescribed in Axelos et al., Mol. Gen. Genet. 219:106-112, 1989),operably linked to the Arabidopsis EPSPS chloroplast transit peptide(TS-At.EPSPS:CTP2, Klee et al., Mol. Gen. Genet. 210:47-442, 1987),operably linked to a glyphosate tolerant5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) from Agrobacteriumsp. strain CP4 (aroA:CP4, U.S. Pat. No. 5,633,435), operably linked tothe 3′ termination region from pea ribulose 1,5-bisphosphate carboxylaseE9 (T-Ps.RbcS2:E9, Coruzzi, et al., EMBO J. 3:1671-1679, 1984), and asecond expression cassette comprising the CaMV35S-Act8 promoterincluding the first intron of the ActB gene (SEQ ID NO:29, U.S. Pat. No.6,462,258) operably connected to an Arabidopsis EPSPS chloroplasttransit peptide (TS-At.EPSPS:CTP2), operably connected to a glyphosatetolerant 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) fromAgrobacterium sp. strain CP4 (aroA:CP4, U.S. Pat. No. 5,633,435, hereinincorporated by reference in its entirety), operably linked to the 3′termination region from pea ribulose 1,5-bisphosphate carboxylase E9.

The following examples are included to demonstrate examples of certainpreferred embodiments of the invention. It should be appreciated bythose of skill in the art that the techniques disclosed in the examplesthat follow represent approaches the inventors have found function wellin the practice of the invention, and thus can be considered toconstitute examples of preferred modes for its practice. However, thoseof skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentsthat are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

EXAMPLES Example 1

The transgenic cotton event MON 88913 was generated by anAgrobacterium-mediated transformation of cotton cells with a DNAfragment derived from pMON51915 (FIG. 1). The plant transformationconstruct, pMON51915 was mated into Agrobacterium using a triparentalmating procedure (Ditta et al., Proc. Natl. Acad. Sci. 77:7347-7351,1980). Cotton cell transformation with transgenes can be performed usingmethods described, e.g., in U.S. Pat. No. 5,004,863, U.S. Pat. No.5,159,135, and U.S. Pat. No. 5,518,908, herein incorporated by referencein their entirety. Cotton transformation is performed essentially asdescribed in WO/0036911 or as described in U.S. Pat. No. 5,846,797,herein incorporated by reference in its entirety. A modification ofthese methods can include, but is not limited to the following example.Coker 130 seed is surface sterilized and germinated in the dark.Hypocotyl explants are cut from the germinated seedlings to lengths ofabout 1-1.5 centimeter. Agrobacterium tumefaciens strain ABI transformedto contain pMON51915 is grown in Luria broth without antibiotics for 16hours at 28° C., then diluted to approximately 2×10⁸ bacteria/milliter(ml). The hypotocyl explant is submersed in the Agrobacterium inoculumfor 2-5 minutes, then co-cultivated for about 45 hours on MS+1.9 mg/lKNO3+3% glucose (TRM), 30 explants per plate, 24 C, in the dark. Theexplants are transferred to TRM containing 150 mg/l cefotaxime and 300EM glyphosate for four culture periods, each period for approximatelysix weeks. Embryogenic calli is segregated from the primary explant atthe end of 3rd or 4th culture periods and placed onto same medium. Theembryogenic calli are subcultured once by briefly suspending in liquidTRM+3% glucose, followed by pouring suspension onto ‘TRM’+150 mg/lcefotaxime+300 μM glyphosate plates. The somatic embryos are harvested3-8 weeks after the liquid subculture, then grown on Stewart and Hsumedia with 0.5% glucose. Plantlets derived from the somatic embryos arematured to about 4-7 cm (3-6 leaves) in Magenta boxes with Stewart & Hsumodified with 40 mM NO3/lOmM NH4+2% sucrose. These plants are thentransplanted to potting soil, 4″ pots, 100% humidity, 16 hours of lightper day, for 4-6 days, followed by 50% humidity 5-10 days.

The DNA fragment of pMON51915 contains two transgene expressioncassettes inserted into the genome of MON 88913 (FIG. 2) thatcollectively confer glyphosate tolerance to MON 88913 and progenythereof.

The MON 88913 plant and seed has regenerable parts. The regenerableparts of the seed include, but are not limited to the embryo, thecotyledon, and the shoot or root meristem. The regenerable parts of theplant include, but are not limited to the leaves, the petiole, thehypocotyl, stem sections, and apical and root meristems. The inventionalso includes plant parts of cotton event MON 88913 that include, butare not limited to pollen, ovule, flowers, bolls, lint, shoots, roots,and leaves. The invention also includes extractable components of MON88913 seed that include, but are not limited to protein, meal, flour,hulls, oil, and linter.

Example 2

The glyphosate tolerant cotton event MON 88913 was selected from manytransgenic cotton events for tolerance to glyphosate vegetative andreproductive injury. The successful production of a commercial qualitytransgenic event currently requires producing a large number oftransgenic events. In the present invention, MON 88913 was one eventamong approximately 1000 R0 events that had been transformed with manydifferent DNA constructs that included pMON51915. The MON 88913 eventwas selected from the many events by a series of molecular analysis andglyphosate tolerance screens.

The events were screened in a greenhouse glyphosate tolerance test, theplants being scored for vegetative and reproductive tolerance. Fifteento twenty-five R₁ seeds from each event were planted in 15 cell trayswith Metro-Mix 350 growing medium, which contains a combination of peat,vermiculite, nutrients, wetting agents, and processed bark and ash.Additional fertilizers included in the medium were Osmocote 14-14-14,Osmocote Plus 15-9-12, and MicroMax micronutrients. All plants weregrown in a greenhouse. The average daytime temperature during thegrowing season was 32 degrees Celsius (° C.), while the average nighttemperature was 24° C. The photoperiod was set at 16 hours of light andeight hours of dark, with maximum light intensity. The average relativehumidity during the growth cycle was 45 percent. The plants were thensprayed at the 4 and 8-leaf stages sequentially with 48 oz/A (oz=ounces,A=acre) of Roundup Ultra® (glyphosate containing herbicide). Seven daysafter the 4-leaf glyphosate application, the plants were scored forvegetative damage and segregation of the glyphosate tolerant phenotypewas collected. These data were used to confirm that the event transgeneinsert was performing as a single dominant gene, adhering to Mendeliangenetic models. Events with good vegetative tolerance were subsequentlytransplanted in 10-inch pots with the same Metro-Mix 350 growing mediumdescribed above and grown to maturity. Plants were treated with Pix Plus(BASF, Research Triangle Park, N.C.) as needed to regulate the plantheight. At three months post-planting, the plants were mapped for bollretention on the first fruiting positions of the first five fruitingbranches. The maximum value for retention for this plant map is 5 (fivebolls retained). This provided a quick, indirect measure of plantfertility. With this greenhouse screen, the average retention for thecurrent commercial event (RR cotton 1445) is less than 1.0. Events withan average boll retention value greater than or equal to three wereharvested and advanced for further event selection. Events that haveboll retention values greater than or equal to two have value as newglyphosate tolerant plant selections.

Events that met the Roundup® Ultra vegetative and reproductive tolerancecriteria were analyzed for copy number via Southern blot analysis.Single copy events that showed good tolerance in initial greenhouseexperiments were further characterized in 1) additional greenhousetolerance tests at higher glyphosate rates, 2) replicated field trials,and with 3) additional molecular screens. The greenhouse tolerance testswere conducted using homozygous plants. All of the experiments containedthe current commercial Roundup Ready® cotton event 1445 event(commercial standard) for comparison. Seed were planted in 15 cell traysand treated with 64 oz/A Roundup Ultra® at the 4-leaf stage and 96 oz/Aat the 8-leaf stage. The plants were then transplanted to 10-inch potsand four plants were mapped at mid-season on the first fruitingpositions of the first five fruiting branches. End of the season datawere also collected on all events and included seed cotton weight,number of bolls, boll size, and boll retention.

Field testing was used to select the event that showed best growthrates, fruit retention, and yield. The field trials were arranged in arandomized split plot design with three replications and threetreatments. The events were planted in two row, 30-foot plots. Thetreatments consisted of unsprayed, 64 oz/A (1.5 lb ae/A) Roundup Ultra®at the 4, 6, 10, 14-node stages, and 96 oz/A (2.25 lb ae/A) RoundupUltra® at the 4, 6, 10, 14-node stages. A mid-season plant map wascompleted on ten plants per plot. Boll retention data was collected forthe first and second fruiting positions of the first five fruiting nodesthat provides a boll retention value scale of 0-10. A plant with a bollretention value equal to or greater than 3 on the 0-10 scale has valueas a new glyphosate tolerant plant selection.

Field testing (10 locations) comparing the yield of cotton lint(pounds/acre, lb/A) from MON 88913 and RR cotton 1445 showed that MON88913 provided substantial protection against glyphosate (pounds of acidequivalent/acre, lb ae/A) effects on yield (Table 1). Yield is a measureof boll retention, cotton plants engineered for glyphosate tolerancethat retain a substantial number of bolls in the first and secondfruiting positions will maintain a yield advantage over cotton plantsthat are not as glyphosate tolerant. An effective dose of a glyphosatecontaining herbicide to control weeds in a field of MON 88913 comprisesabout 4 oz/A and may exceed 128 oz/A depending on the species of weed tobe controlled and the stage of weed development. Glyphosate can be mixedwith other herbicides to enhance the herbicidal activity against certainweed species.

TABLE 1 Comparison of lint yield of MON 88913 and 1445 after glyphosatetreatment. Glyphosate Yield (lb/A) of 10 Locations treatment 0 lb ae/A1.5 lb ae/A 2.25 lb ae/A 1445 2421.84 1044.19 831.47 MON 88913 2551.582587.61 2412.3

Example 3

Cotton genomic DNA for all PCR reactions and Southern blot analyses wasisolated using a CTAB procedure (Rogers et al., Plant Mol. Biol.5:69-76, 1985) or Dneasy™ 96 Plant Kit (Cat. #69181, Qiagen Inc.,Valencia, Calif.) following the manufacturers instructions. Leaf tissuewas collected from plants at the 2-4-leaf stage. The smallest trueleaves were collected from each plant and immediately frozen on dry ice.DNA was extracted using, e.g., the following method. The tissue wasground using plastic beads with liquid nitrogen. Five ml of extractionbuffer was added to 0.75 gram (g) of tissue and incubated at 55° C. for45 minutes. The CTAB extraction buffer consisted, of 100 mM Tris pH8.0,1.4M NaCl, 20 mM EDTA, 2% CTAB with the addition of 5 μl (microliter) ofbeta-mercaptoethanol, 5 μl of RNase and 1% PVPP. The samples were thenextracted with an equal volume of chloroform (5 ml) and then centrifugedat 3700 RPM for 15 minutes at room temperature. The aqueous phase wastransferred to a new tube and the DNA was precipitated with an equalvolume of isopropanol. After centrifugation at 3700 RPM for 15 minutes,the pellets were washed with 70% ethanol, air dried, and resuspended in250 μl of water.

Cotton genomic DNA adjacent to the transgene insertion was obtained forthe MON 88913 event utilizing TAIL-PCR (Liu et al., Plant Journal 8:457-463, 1995). Extension of the genomic DNA was conducted using theGenomeWalker kit (CloneTech Laboratories, Palo Alto, Calif.) followingthe manufacture's protocol. Briefly, the DNA (˜5 μg) isolated utilizingthe CTAB protocol previously described, was digested with variousrestriction endonucleases (EcoRV, Sca1) at 37° C. overnight in a totalvolume of 100 μl. The restriction endonucleases were removed withQIAquick PCR Purification columns (cat #28104, Qiagen, Inc.). Theligation of adaptor molecules were those that were described in themanufacture's protocol. DNA was amplified using FMV-1 primer (SEQ IDNO:5) with the AP1 primer (CloneTech Laboratories) for the primaryreaction and nested FMV-2 primer (SEQ ID NO:6) with the AP2 primer(CloneTech Laboratories) for the secondary reaction.

The 3′ transgene/genomic DNA of the MON 88913 was isolated utilizinginverse PCR. Total genomic DNA (˜10 μg) was digested with threerestriction enzymes; BclI, NcoI, and HindIII. The QIAquick PCRPurification columns were used to purify the DNA after digestingovernight at 37° C. The DNA was eluted from the columns with 50 μl ofwater and then diluted to 1 ml. The diluted eluate (85 μl) was combinedwith 10 μl of buffer (10×) and 5 μl of T4 Ligase to circularize thefragments. After an overnight incubation at 16° C., the ligase was heatinactivated at 70° C. The samples were amplified by PCR with a series ofnested primers. The primer combinations for PCR included: primary pair8099-E9-1/E9-2 (SEQ ID NO:7/SEQ ID NO:8) for BclI and NcoI samples andprimer pair 8099-E9-1/Act8 rev (SEQ ID NO:7/SEQ ID NO:9) for the HindIIIsample; primer pair 8099-E9-2/E9-1 (SEQ ID NO:10/SEQ ID NO:11) for BclIand NcoI samples; primer pair 8099-E9-2/Act8 (SEQ ID NO:10/SEQ ID NO:12)for the HindIII sample; primer pair 8099-E9-3/E9-1 (SEQ ID NO:13/SEQ IDNO:11) for BclI and NcoI samples and 8099-E9-3/Act8 (SEQ ID NO:13/SEQ IDNO:12) for the HindIII sample. The conditions for the PCR included:primary PCR=7 cycles of 94° C. for 2 seconds, 72° C. for 10 minutes; 37cycles of 94° C. for 2 seconds, 67° C. for 10 minutes; 1 cycle of 67° C.for 10 minutes; secondary and tertiary PCR=5 cycles of 94° C. for 2seconds, 72° C. for 10 minutes; 24 cycles of 94° C. for 2 seconds, 67°C. for 10 minutes; 1 cycle of 67° C. for 10 minutes.

Alternatively, DNA amplification by PCR of the 3′ end of the MON 88913event can be performed with conditions that include: 7 cycles of 94° C.for 25 seconds, 72° C. for 3 minutes; 37 cycles of 94° C. for 25seconds, 67° C. for 3 minutes; 1 cycle of 67° C. for 7 minutes. Allsubsequent amplifications conducted with the following conditions: 7cycles of 94° C. for 2 seconds, 72° C. for 4 minutes; 37 cycles of 94°C. for 2 seconds, 67° C. for 4 minutes; 1 cycle of 67° C. for 7 minutes.All amplicons are visualized on 0.8% agarose gels stained with ethidiumbromide. The DNA is prepared for sequencing either by purifying the PCRsamples directly with the QIAquick PCR Purification kit (cat#28104,Qiagen Inc.) or by extracting the appropriate fragment from the gel andusing the QIAquick Gel Extraction kit (cat #28704, Qiagen Inc.).

A series of DNA primers were designed to sequence the transgene insertand the adjacent flanking genomic regions of the MON 88913. DNA primerswere designed that allowed amplification of the entire transgene andgenomic flanking regions by five overlapping fragments. Unique primerswere designed to allow amplification of eachEPSPS-CTP2/aroA-CP4/RbcS2:E9 region separately. For all fragments usedin sequencing, the amplifications were performed in triplicate. The DNAprimer pair combinations used as sequencing primers for the 5′transgene/genomic region (SEQ ID NO:14 and SEQ ID NO:15), 3′transgene/genomic region (SEQ ID NO:16 and SEQ ID NO:17) and insertgenetic elements (SEQ ID NO:18 and SEQ ID NO:11; SEQ ID NO:19 and SEQ IDNO:15; SEQ ID NO:20 and SEQ ID NO:11). Total genomic DNA was used forall PCR reactions. All amplicons were visualized on 0.8% agarose gelsstained with ethidium bromide. The DNA was prepared for sequencingeither by purifying the PCR samples directly with the QIAquick PCRPurification kit or by extracting the appropriate fragment from the geland using the QIAquick Gel Extraction kit. The DNA sequence was producedusing DNA sequence analysis equipment (ABI Prism™ 377, PE Biosystems,Foster City, Calif.) and DNASTAR sequence analysis software (DNASTARInc., Madison, Wis.).

The DNA fragments from the flanking regions of MON 88913transgene/genomic insert were subcloned using a TOPO TA Cloning® kit(Invitrogen). The DNA sequence of the 5′ transgene/genomic region isshown in FIG. 4 and the DNA sequence of the 3′ transgene/genomic regionis shown in FIG. 5. In the DNA sequence shown in FIGS. 4 and 5, thetransgene insert sequence is in italics.

Example 4

DNA event primer pairs are used to produce an amplicon diagnostic forcotton event MON 88913 genome. Amplicons diagnostic for MON 88913 genomecomprise at least one junction sequence, SEQ ID NO:1 or SEQ ID NO:2.Event primer pairs that will produce a diagnostic amplicon for MON88913, in which the primer pairs include, but are not limited to SEQ IDNO:14 and SEQ ID NO:15 for the 5′ amplicon sequence, and SEQ ID NO:16and SEQ ID NO:17 for the 3′ amplicon when used in the protocol outlinedin Table 2. In addition to these primer pairs, any primer pair,homologous or complementary to SEQ ID NO:3 or SEQ ID NO:4, that in a DNAamplification reaction produces an amplicon diagnostic for MON 88913genome is an aspect of the present invention. Any single isolated DNApolynucleotide primer molecule comprising at least 11 contiguousnucleotides of SEQ ID NO:3, or its complement that is useful in a DNAamplification method to produce an amplicon diagnostic for MON 88913 isan aspect of the invention. Any single isolated DNA polynucleotideprimer molecule comprising at least 11 contiguous nucleotides of SEQ IDNO:4, or its complement that is useful in a DNA amplification method toproduce an amplicon diagnostic for MON 88913 is an aspect of theinvention. An example of the amplification conditions for this analysisis illustrated in Table 2 and Table 3, however, any modification ofthese methods that use DNA primers homologous or complementary to SEQ IDNO:3 or SEQ ID NO:4 or DNA sequences of the genetic elements containedin the transgene insert of MON 88913 that produce an amplicon diagnosticfor MON 88913, is within the ordinary skill of the art. A diagnosticamplicon comprises a DNA molecule homologous or complementary to atleast one transgene/genomic junction DNA (SEQ ID NO:1 or SEQ ID NO:2) orsubstantial portion thereof.

An analysis for event MON 88913 plant tissue sample should include apositive tissue control from event MON 88913, a negative control from acotton plant that is not event MON 88913, and a negative control thatcontains no cotton genomic DNA. Additional primer sequences can beselected from SEQ ID NO:3 and SEQ ID NO:4 by those skilled in the art ofDNA amplification methods, and conditions selected for the production ofan amplicon by the methods shown in Table 2 and Table 3 may differ, butresult in an amplicon diagnostic for event MON 88913. The use of theseDNA primer sequences with modifications to the methods of Table 2 and 3are within the scope of the invention. The amplicon produced by at leastone DNA primer sequence derived from SEQ ID NO:3 or SEQ ID NO:4 that isdiagnostic for MON 88913 is an aspect of the invention.

DNA detection kits that contain at least one DNA primer derived from SEQID NO:3 or SEQ ID NO:4 that when used in a DNA amplification methodproduces a diagnostic amplicon for MON 88913 is an aspect of theinvention. The amplicon produced by at least one primer sequence derivedfrom any of the genetic elements of pMON51915 that is diagnostic for MON88913 is an aspect of the invention. A cotton plant or seed, wherein itsgenome will produce an amplicon comprising SEQ ID NO:1 or SEQ ID NO:2when tested in a DNA amplification method is an aspect of the presentinvention. The assay for the MON 88913 amplicon can be performed byusing a Stratagene Robocycler, MJ Engine, Perkin-Elmer 9700, orEppendorf Mastercycler Gradient thermocycler as shown in Table 3, or bymethods and apparatus known to those skilled in the art.

TABLE 2 PCR procedure and reaction mixture conditions for theidentification of MON 88913 5′ transgene insert/genomic junction region.Step Reagent Amount Comments 1 Nuclease-free water add to final volumeof 20 μl — 2 10X reaction buffer 2.0 μl 1X final concentration of (withMgCl₂) buffer, 1.5 mM final concentration of MgCl₂ 3 10 mM solution ofdATP, 0.4 μl 200 μM final dCTP, dGTP, and dTTP concentration of eachdNTP 4 event primer (SEQ ID 0.4 μl 0.2 μM final NO: 14) (resuspended in1X concentration TE buffer or nuclease-free water to a concentration of10 μM) 5 event primer (SEQ ID 0.4 μl 0.2 μM final NO: 15) concentration(resuspended in 1X TE buffer or nuclease-free water to a concentrationof 10 μM) 6 RNase, DNase free (500 0.1 μl 50 ng/reaction ng/μl) 7 REDTaqDNA polymerase 1.0 μl (recommended to 1 unit/reaction (1 unit/μl) switchpipets prior to next step) 8 Extracted DNA (template): — Samples to beanalyzed individual leaves 10-200 ng of genomic DNA pooled leaves 200 ngof genomic DNA (maximum of 50 leaves/pool) Negative control 50 ng ofcotton genomic DNA (not MON 88913) Negative control no template DNAPositive control 50 ng of MON 88913 genomic DNA 9 Gently mix and add 1-2drops of mineral oil on top of each reaction.

TABLE 3 Suggested PCR parameters for different thermocyclers. Proceedwith the DNA amplification in a Stratagene Robocycler, MJ Engine,Perkin-Elmer 9700, or Eppendorf Mastercycler Gradient thermocycler usingthe following cycling parameters. The MJ Engine or EppendorfMastercycler Gradient thermocycler should be run in the calculated mode.Run the Perkin-Elmer 9700 thermocycler with the ramp speed set atmaximum. Cycle No. Settings: Stratagene Robocycler 1 94° C. 3 minutes 3894° C. 1 minute 60° C. 1 minute 72° C. 1 minute and 30 seconds 1 72° C.10 minutes Settings: MJ Engine or Perkin-Elmer 9700 1 94° C. 3 minutes38 94° C. 10 seconds 60° C. 30 seconds 72° C. 1 minute 1 72° C. 10minutes Settings: Eppendorf Mastercycler Gradient 1 94° C. 3 minutes 3894° C. 15 seconds 60° C. 15 seconds 72° C. 1 minute and 30 seconds 1 72°C. 10 minutes

Example 5

MON 88913 genomic DNA and control cotton genomic DNA (˜15 μg of each) isdigested with various restriction enzymes (140U) in a total volume of150 μincluding 15111 of the corresponding manufacturer's buffer (NEB,Beverely, Mass.). Restriction endonucleases, e.g., BglI, BamHI, NcoI,HindIII, and BcII, are used in the Southern analysis of MON 88913.Endonuclease digests are performed at the appropriate temperature for atleast 6 hours. After incubating, the DNA is precipitated with 3M sodiumacetate and 2.5 volumes of ethanol. Subsequently, the DNA is washed with70% ethanol, dried, and resuspended in 40 μl of TBE. Loading buffer(0.2×) is added to the samples and then electrophoresis conducted onagarose gels (0.8%) for 16-18 hours at 30 volts. The gels are stainedwith ethidium-bromide, then treated with a depurination solution (0.125NHCL) for 10 minutes, with a denaturing solution (0.5M sodium hydroxide,1.5M sodium chloride) for 30 minutes, and finally with a neutralizingsolution (0.5M Trizma base, 1.5M sodium chloride) for 30 minutes. TheDNA is transferred to Hybond-N membrane (Amersham Pharmacia Biotech,Buckinghamshire, England) using a Turboblotter (Schleicher and Schuell,Dassel, Germany) for 4-6 hours and then fixed to the membrane using a UVlight.

Membranes are prehybridized with 20 mls of DIG Easy Hyb solution (RocheMolecular Biochemicals, Indianapolis, Ind.; cat. #1603558) for 2-4 hoursat 45° C. Radioactive DNA probes (³²P dCTP) homologous or complementaryto SEQ ID NO:1, or SEQ ID NO:2, or SEQ ID NO:3, or SEQ ID NO:4, or aportion thereof are made using a Radprime DNA Labeling kit (Invitrogen,Carlsbad, Calif.; cat. #18428-011). Unincorporated nucleotides areremoved using sephadex G-50 columns (Invitrogen). The prehybridizationsolution is replaced with 10 mls of pre-warmed DIG Easy Hyb solutioncontaining the denatured probe to a final concentration of 1 millioncounts per ml. The blots are hybridized at 45° C. for 16-18 hours.

Blots are washed with a low stringency solution (5×SSC, 0.1×SDS) at 45°C. and then repeatedly washed with a higher stringency solution(0.1×SSC, 0.1% SDS) at 65° C. The blots are exposed to a phosphor screen(Amersham Biosciences, Piscataway, N.J.) for >2 hours and the exposureread using a Data Storm 860 machine (Amersham Biosciences).

Example 6

The methods used to identify heterozygous from homozygous cotton progenycontaining event MON 88913 are described in a zygosity assay for whichexamples of conditions are described in Table 4 and Table 5. The DNAprimers used in the zygosity assay are primer SQ1099 (SEQ ID NO:21),SQ1100 (SEQ ID NO:22), SQ1353 (SEQ ID NO:23), 6FAM™ labeled primer (SEQID NO:24,), and VIC™ labeled primer (SEQ ID NO:25), 6FAM and VIC areflorescent dye products of Applied Biosystems (Foster City, Calif.)attached to the DNA primer.

SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23 when used in these reactionmethods produce a DNA amplicon for non-transgenic cotton, two DNAamplicons for heterozygous cotton containing event MON 88913, and a DNAamplicon for homozygous MON 88913 cotton that is distinct from any othernon-MON 88913 cotton. The controls for this analysis should include apositive control from homozygous and heterozygous cotton containingevent MON 88913 DNA, a negative control from non-transgenic cotton, anda negative control that contains no template DNA. This assay isoptimized for use with a Stratagene Robocycler, MJ Engine, Perkin-Elmer9700, or Eppendorf Mastercycler Gradient thermocycler. Other methods andapparatus known to those skilled in the art that produce amplicons thatidentify the zygosity of the progeny of crosses made with MON 88913cotton plants is within the skill of the art.

TABLE 4 Zygosity assay reaction solutions Step Reagent Amount Comments 1Nuclease-free water add to 10 μl final volume — 2 2X Universal MasterMix (Applied 5 μl 1 X final Biosystems cat. # 4304437) concentration 3Primers SQ1099, SQ1100, SQ1353 0.5 μl 0.25 μM final (resuspended innuclease-free water concentration to a concentration of 20 μM) 4 Primer6FAM ™ (resuspended in 0.2 μl 0.4 μM final nuclease-free water to aconcentration concentration of 10 μM) 5 Primer VIC ™ (resuspended in 0.2μl 0.15 μM final nuclease-free water to a concentration concentration of10 μM) 6 REDTaq DNA polymerase 1.0 μl (recommended to 1 unit/reaction (1unit/μl) switch pipets prior to next step) 7 Extracted DNA (template):3.0 μl Diluted in water Samples to be analyzed (individual 4-80 ng ofgenomic leaves) DNA Negative control 4 ng of non-transgenic cottongenomic DNA Negative control no DNA template (solution in which DNA wasresuspended) Positive control 4 ng of genomic DNA from known event MON88913 heterozygous cotton Positive control 4 ng of genomic DNA fromknown event MON 88913 homozygous cotton 8 Gently mix, add 1-2 drops ofmineral oil on top of each reaction.

TABLE 5 Zygosity assay thermocycler conditions Proceed with the DNAamplificaition in a Stratagene Robocycler, MJ Engine, Perkin-Elmer 9700,or Eppendorf Mastercycler Gradient thermocycler using the followingcycling parameters. When running the PCR in the Eppendorf MastercyclerGradient or MJ Engine, the thermocycler should be run in the calculatedmode. When running the PCR in the Perkin-Elmer 9700, run thethermocycler with the ramp speed set at maximum. Cycle No. Settings:Stratagene Robocycler 1 94° C. 3 minutes 38 94° C. 1 minute 60° C. 1minute 72° C. 1 minute and 30 seconds 1 72° C. 10 minutes Settings: MJEngine or Perkin-Elmer 9700 1 94° C. 3 minutes 38 94° C. 30 seconds 60°C. 30 seconds 72° C. 1 minute and 30 seconds 1 72° C. 10 minutesSettings: Eppendorf Mastercycler Gradient 1 94° C. 3 minutes 38 94° C.15 seconds 60° C. 15 seconds 72° C. 1 minute and 30 seconds 1 72° C. 10minutes

Example 7

Analysis of cotton genomic DNA samples was conducted using an endpointTaqman® method. The production of amplicons diagnostic for MON 88913genomic DNA were produced by using a primer set A that included eventprimers: SEQ ID NO:21, SEQ ID NO:22, and 6-FAM probe SEQ ID NO:24; and aprimer set B that included event primers: SEQ ID NO:26, SEQ ID NO:27,and 6-FAM probe SEQ ID NO:28. The method uses a 96-well or 384-wellformat and an Applied Biosystems GeneAmp PCR System 9700 or MJ ResearchDNA Engine PT-225. DNA extracted from cotton tissue samples aspreviously described should be within the range of 5-10 ng per PCRreaction. Each reaction contains a final volume of 10 μl consisting of0.5 μl of equal concentration of the event primers (20 μM), 5.0 μl of 2×universal master mix, 0.41 of the 6-FAM probe (10 μM), 3 μl DNA sample(5-10 ng) and water to 10 μl. The thermal cycler parameters are 1 cycle50° C. for 2 minutes, 1 cycle 95° C. for 10 minutes, 10 cycles at 95° C.for 15 seconds, 64° C. for 1 minute then −1° C./cycle, 30 cycles 95° C.for 15 seconds, 54° C. for 1 minute, then hold at 10° C. The ampliconproduction was determined by a microplate reader, e.g., a TECAN Safire(Durham, N.C.) using the conditions described by the manufacturer. Adata analysis program (TaqPro™) was used to score the production of thelabeled amplicon. Other equipment and analysis methods known in the artof DNA detection can be used to detect the amplicons of the presentinvention.

A deposit of Monsanto Technology LLC, cotton MON 88913 seed disclosedabove and recited in the claims, has been made under the Budapest Treatywith the American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, Va. 20110. The ATCC accession number is PTA-4854.The deposit will be maintained in the depository for a period of 30years, or 5 years after the last request, or for the effective life ofthe patent, whichever is longer, and will be replaced as necessaryduring that period.

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims.

All publications and published patent documents cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

1-6. (canceled)
 7. An isolated DNA polynucleotide primer moleculecomprising at least 11 contiguous nucleotides of SEQ ID NO:3 or SEQ IDNO:4, or its complement that is useful in a DNA amplification method toproduce an amplicon comprising SEQ ID NO:1 or SEQ ID NO:2 diagnostic forcotton event MON
 88913. 8. The isolated DNA polynucleotide primermolecule of claim 7, comprising at least 11 contiguous nucleotides ofSEQ ID NO:4, or its complement that is useful in a DNA amplificationmethod to produce an amplicon comprising SEQ ID NO:2 diagnostic forcotton event MON
 88913. 9. A DNA detection kit comprising the primermolecule according to claim
 7. 10-11. (canceled)
 12. A method ofdetecting the presence of DNA corresponding to cotton event MON 88913 ina sample, the method comprising: (a) contacting the sample comprisingDNA with a DNA primer set comprising a pair of DNA polynucleotide primermolecules according to claim 7 that, when used in a nucleic acidamplification reaction with genomic DNA from the cotton event MON 88913,produces a diagnostic amplicon comprising SEQ ID NO:1 or SEQ ID NO:2;and (b) performing a nucleic acid amplification reaction, therebyproducing the diagnostic amplicon; and (c) detecting the diagnosticamplicon.
 13. The method of claim 12, wherein said primer set comprisesa primer selected from the group consisting of SEQ ID NO:21, SEQ IDNO:22 and SEQ ID NO:24.
 14. The method of claim 12, wherein said primerset comprises a primer selected from the group consisting of SEQ IDNO:26, SEQ ID NO:27 and SEQ ID NO:28. 15-16. (canceled)
 17. A method ofdetermining the zygosity of the progeny of cotton event MON 88913comprising: (a) contacting a sample comprising cotton DNA with a set ofprimers according to claim 7 that, when used in a nucleic-acidamplification reaction with genomic DNA comprising cotton event MON88913, produces a first amplicon that is diagnostic for cotton event MON88913; (b) performing a nucleic acid amplification reaction, therebyproducing the first amplicon; (c) detecting the first amplicon; (d)contacting the sample comprising cotton DNA with said set of primersthat when used in a nucleic-acid amplification reaction with genomic DNAfrom cotton plants comprising event MON 88913, produces a secondamplicon comprising the native cotton genomic DNA homologous to thecotton genomic region of a transgene insertion identified as cottonevent MON 88913; (e) performing a nucleic acid amplification reaction,thereby producing the second amplicon; (f) detecting the secondamplicon; and (g) comparing the first and second amplicons in a sample,wherein the presence of both amplicons indicates the sample isheterozygous for the transgene insertion.
 18. A method of determiningthe zygosity of the progeny of cotton event MON 88913 comprising: (a)contacting a sample comprising cotton DNA with a set of primersaccording to claim 7, (b) performing a nucleic acid amplificationreaction; and (c) detecting the products of the reaction.
 19. (canceled)20. The method of claim 17, wherein the primer set comprises a primerselected from the group consisting of SEQ ID NO:21, SEQ ID NO:22 and SEQID NO:23, SEQ ID NO:24, and SEQ ID NO:25.
 21. The method of claim 18,wherein the primer set comprises a primer selected from the groupconsisting of SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23, SEQ ID NO:24,and SEQ ID NO:25.
 22. A pair of primer molecules according to claim 7.23. The isolated DNA polynucleotide primer molecule of claim 7,comprising at least 11 contiguous nucleotides of SEQ ID NO:3, or itscomplement that is useful in a DNA amplification method to produce anamplicon comprising SEQ ID NO:1 diagnostic for cotton event MON 88913.